nitric oxide signalling in plants

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Review

Key words:

Abscisic acid (ABA); cyclic ADP ribose and cyclic GMP; cyclic nucleotide-gated ion channels; nitric oxide synthase; nitrate reductase; plant–pathogen interactions; signal transduction; stomata.

Blackwell Publishing Ltd.Oxford, UKNPHNew Phytologist1469-8137Trustees of New Phytologist 2003159Tansley reviewTansley reviewTansley review

Tansley review

Nitric oxide signalling in plants

Steven J. Neill, Radhika Desikan and John T. Hancock

Centre for Research in Plant Science, University of the West of England (UWE), Bristol, Coldharbour

Lane, Bristol BS16 1QY, UK

Contents

Summary 111. Introduction 122. Why does NO make a good signal? 123. NO biosynthesis 134. NO biology 17

5. NO signal transduction 236. Conclusion 30

Acknowledgements 31References 31

Author for correspondence:

Steven J. Neill Tel: +44 1173442149 Fax: +44 1173442904 Email: [email protected]

Received:

11 December 2002

Accepted:

19 March 2003

doi: 10.1046/j.1469-8137.2003.00804.x

Summary

Recently nitric oxide (NO) has emerged as a key signalling molecule in plants. Herewe review the potential sources of endogenous NO, outline the biological processeslikely to be mediated by NO, and discuss the downstream signalling processes bywhich NO exerts its cellular effects. It will be important to develop methods to quan-tify intracellular NO synthesis and release. Clasification of the biosynthetic origins ofNO is also required. NO can be synthesised from nitrite via nitrate reductase (NR)and although biochemical and immunological data indicate the presence ofenzyme(s) similar to mammalian nitric oxide synthase (NOS), no NOS genes havebeen identified. NO can induce various processes in plants, including the expressionof defence-related genes and programmed cell death (PCD), stomatal closure, seedgermination and root development. Intracellular signalling responses to NO involvegeneration of cGMP, cADPR and elevation of cytosolic calcium, but in many cases,the precise biochemical and cellular nature of these responses has not been detailed.Research priorities here must be the reliable quantification of downstream signallingmolecules in NO-responsive cells, and cloning and manipulation of the enzymesresponsible for synthesis and degradation of these molecules.

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1. Introduction

Nitric oxide, NO, is a small, water and lipid soluble gas thatin recent years has emerged as a major signalling molecule ofancient origin and ubiquitous importance (Durner

et al

.,1999). In 1992 it was named ‘Molecule of the Year’ by

Science

(Koshland, 1992) and since then there has been a hugenumber of studies on NO biology. NO emission from plantsand its effects on plant growth were described in the early1970s (Anderson & Mansfield, 1979; Klepper, 1979).However, research on NO and plant signalling was mainlyrestricted to a few ‘pioneers’ such as Leshem (Leshem &Haramaty, 1996) and Lamattina (Laxalt

et al

., 1997) until thetwo landmark publications in 1998 describing NO as a plantdefence signal (Delledonne

et al

., 1998; Durner

et al

., 1998).Since then, studies on NO and plant biology have increaseddramatically, with some of this work being reviewed relativelyrecently (Durner & Klessig, 1999; Beligni & Lamattina,2001; Wendehenne

et al

., 2001; Neill

et al

., 2002b). In thisarticle, we attempt to describe our current understanding ofNO biochemistry in plants, highlight the growing number ofbiological processes likely to involve NO signalling, andoutline the signalling mechanisms by which NO may exert itscellular effects.

2. Why does NO make a good signal?

Nitric oxide (NO

·

) is a gaseous free radical. It contains anunpaired electron in its

π

2

orbital, but remains uncharged.However, because of its free radical nature, it can adopt anenergetically more favourable electron structure by gainingor losing an electron, so that NO can exist as threeinterchangeable species: the radical (NO

·

); the nitrosoniumcation (NO

+

); and the nitroxyl radical (NO

–

) (Stamler

et al

.,1992; Wojtaszek, 2000). NO is sparingly soluble in water(0.047 cm

3

cm

−

3

H

2

O; 20

°

C 1 atm), with the solubilityincreasing in the presence of ferrous salts (Anderson &Mansfield, 1979). Therefore, it is able to move by diffusion inaqueous parts of the cell, such as the cytoplasm, but also movefreely through the lipid phase of membranes. Once produced,it can move from one cell to another or within a cell. How-ever, being a reactive free radical, it has a relatively shorthalf-life, in the order of a few seconds. Typically, NO rapidlyreacts with O

2

to form nitrogen dioxide (NO

2

), and rapidlydegrades to nitrite and nitrate in aqueous solution (Fig. 1). Infact, the formation of nitrites and nitrates is often useddiagnostically to measure the previous presence of NO. Thus,the range of its effects is limited to the cell in which it isgenerated, or to cells in the near neighbourhood.

For a signalling molecule to be effective, it needs to be pro-duced quickly and efficiently on demand, to induce definedeffects within the cell, and to be removed rapidly and effec-tively when no longer required. Alternatively, it is possiblethat signals function in concert. For example, it could be that

continuous NO synthesis is essential for other signalling path-ways to operate, such that removal of NO would be inhibi-tory, even though induction of NO synthesis was not required

per se

. Another potential scenario might be that NO, consti-tutively generated, inhibits a particular cellular process. Anystimulus that inhibited NO production would increase thecellular event by virtue of its inhibition of NO synthesis. Asdetailed below, there are several potential cellular sources ofNO in plants, while its chemical structure enables it to moveand relay a signal, and be removed efficiently, terminating themessage it originally was sent to convey.

NO can also react with other potential signalling mole-cules, that are likely to be produced temporally and spatiallyalongside NO. One such chemical is the free radical superox-ide anion (O

2

. –

) (Pryor & Squadrito, 1995), as depicted inFig. 1. Superoxide can arise from several sources in plants,including electron leakage from mitochondria and chloro-plasts, or from more dedicated sources such as NADPH oxi-dases (Neill

et al

., 2002b,c). Superoxide will readily dismuteto hydrogen peroxide (H

2

O

2

), especially at low pH, or in areaction catalysed by superoxide dismutase (SOD). In fact, ifsuperoxide is produced, the presence of hydrogen peroxidebecomes virtually inevitable. Both superoxide and hydrogenperoxide have been suggested as signalling molecules in plants(Jabs, 1999; Neill

et al

., 2002b). Consequently, if NO reactswith superoxide or H

2

O

2

, this could potentially abrogatesuperoxide/hydrogen peroxide signalling, as shown in Figs 1and 2. The product of the reaction between superoxide andthe nitric oxide radical, or H

2

O

2

and NO

+

, is the ion perox-ynitrite, itself a reactive and destructive compound. Suchinteraction between NO and reactive oxygen species (ROS)has been reported particularly during plant–pathogen inter-actions (Delledonne

et al

., 2001). NO can also react with pro-teins, particularly with thiol side groups, or low molecularweight thiols, as discussed in Section 5.5.

Fig. 1 Some of the reactions of nitric oxide (NO). NO can react with oxygen, and in aqueous solution such reaction will lead to the generation of nitrite and nitrate. NO, either as the radical or NO+ ion, can react with superoxide and hydrogen peroxide, respectively, to produce peroxynitrite. NO can also undergo many other reactions with bio-molecules, not shown here.

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3. NO biosynthesis

There are several potential sources of NO in plants (Fig. 2), andit would seem likely that the importance of each of these to thephysiological production of NO will depend on the species,the cells/tissues, the conditions under which the plants aregrown, and, of course, the signalling pathways active underthose specific conditions. There is evidence for NO produc-tion in plants from nitric oxide synthase-like enzymes, nitratereductase, or nonenzymatic sources. These sources, and theirpotential responses to specific conditions are discussed below,and outlined in Table 1.

3.1

Nitric oxide synthase (NOS)

A family of enzymes, the nitric oxide synthases (NOS), isinstrumental in the generation of NO in animal systems, andthis knowledge has prompted a search for homologousmechanisms in plants. In mammals, there are three well-characterised genes encoding NOS. eNOS, or endothelialNOS, is constitutively produced in a wide variety of cells.nNOS, originally characterised from neuronal tissue, is alsoconstitutively produced. On the other hand, iNOS (alter-nately referred to as mNOS) is an inducible form, expressionbeing increased in the presence of lipopolysaccharide orinterferon. nNOS is the largest of the three forms, with a

relative molecular mass of approximately 160 kDa. eNOS isapproximately 135 kDa, and iNOS approximately 130 kDa(Furchgott, 1995). Mitochondrial NOS (mtNOS) has alsobeen reported (Tatoyan & Ginlivi, 1998).

All NOS isoforms appear to exist

in vivo

as homodimers,with a topology of each subunit having two distinct domains,with proteolytic cleavage releasing two active enzymes. Theoxygenase domain contains the haem and tetrahydrobiopt-erin binding sites (Fig. 3); so far tetrahydrobiopterin has notbeen unambiguously identified in plants. The reductasedomain contains binding sites for FAD and NADPH, whilea calmodulin binding site lies between the oxidase and reduct-ase domains. iNOS is Ca

2

+

−

independent, and permanentlybound to calmodulin, but in the case of nNOS and eNOS thecalmodulin is used as a regulatory module and generation ofNO is Ca

2

+

−

dependent.NOS catalyses the formation of NO from

-arginine, whichundergoes a five electron oxidation to

-citrulline. NADPHand molecular oxygen are essential requirements. Arginine isfirst converted to hydroxyarginine, a nonreleased catalyticintermediate, with the final products being citrulline andnitric oxide (Furchgott, 1995). Activity can thus be assayedusing radiolabelled arginine as a substrate, with the formationof radiolabelled citrulline, although it is worth noting thatarginine can also be converted to citrulline without the pro-duction of NO (Ninnemann & Maier, 1996). It should alsobe noted that mammalian NOS is capable of producingsuperoxide ions (Pou

et al

., 1992), and therefore there is thecapacity for the generation of peroxynitrite from this enzyme.

With the well characterised animal NOS as a model, andthe availability of anti-NOS antibodies raised against the

Fig. 2 The production of nitric oxide (NO). There is evidence for several potential sources of NO in plants, including nitric oxide synthase (NOS), nitrate reductase (NR), xanthine oxidoreductase or nonenzymatic sources. Once generated, NO can induce various effects, or react with reactive oxygen species to generate peroxynitrite. It should be noted that NO can exist in three forms, and although it is implied here that only the radical is biologically active, both the NO+ and NO– may have biological effects.

Fig. 3 The topology of nitric oxide synthase and nitrate reductase. Within the tertiary structures of both nitric oxide synthase (NOS) and nitrate reductase (NR), functional domains can be identified, with similarities to other enzymes.

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Table 1

Sources of nitric oxide (NO) in various plants

Sources of NO Evidence Species (Reference) Comments

Nitric oxide synthase • Activity demonstrated Pea (Barroso

et al

., 1999) • ABA-induced NO synthesis and stomatal closure inhibited by NOS inhibitors in pea(NOS) in extracts Maize (Ribeiro

et al

., 1999) • Mechanical stress in

Arabidopsis

induced NOTobacco (Durner

et al

., 1998) • Bacteria-induced NO production not inhibited by NOS inhibitorsSoybean (Delledonne

et al

., 1998) • No NOS-like sequences have been identified

Lupinus albus

(Cueto

et al

., 1996) • Homology to mammalian NOS might be limited

Mucuna hassjoo

(Ninneman & • Little known about cellular location of NOS in plantsMaier, 1996) • False positives reported, casting doubt on immunological data

• Little evidence correlating NOS activity with NO synthesis in the same species

• NOS activity inhibited by Tobacco (Durner

et al

., 1998)inhibitors of mammalian Soybean (Delledonne

et al

., 1998)NOS Pea (Barroso

et al

., 1999)

Lupinus albus

(Cueto

et al

., 1996)

• NO released reduced by Soybean (Delledonne

et al

., 1998)NOS inhibitors Tobbaco (Foissner

et al

., 2000)Pea (Neill

et al

., 2002a)

Arabidopsis

(Garcês

et al

., 2001)

• Anti-NOS antibodies Pea (Sen & Cheema, 1995; Barroso

et al

., 1999)used to identify plant Wheat (Kuo

et al

., 1995; Sen & Cheema, 1995)proteins Maize (Ribeiro

et al

., 1999)

• Anti-NOS antibodies Pea (Barroso

et al

., 1999)inhibit activity

• NO release not

Arabidopsis

(Clarke

et al

., 2000)inhibited by NOS inhibitors Sunflower (Rockel

et al

., 2002)

Chlamydomonas reinhardtii

(Sakihama

et al

., 2002)

Scenedesmus obliquus

(Mallick

et al

., 2000)

Nitrate reductase •

In vitro

production of (Yamasaki & Sakihama, 2000) • NR-mediated NO synthesis required for ABA-induced stomatal closure in

Arabidopsis

(NR) NO by NR Maize (Rockel

et al

., 2002) • Important to determine nitrite and nitrate concentrations in cells

• In vivo

production of Sunflower (Rockel

et al

., 2002) • Sub-cellular localisation of NR needs to be determinedNO by NR Spinach (Rockel

et al

., 2002) • Need to measure NR activities altering following treatments

Arabidopsis

(Desikan

et al

., 2002)

•

NR-deficient mutants have reduced NO generation compared to wild type plants

Soybean (Dean & Harper, 1986)

Arabidopsis

(Desikan

et al

., 2002)

Chlamydomonas reinhardtii

(Sakihama

et al

., 2002)

Xanthine • NO shown to be produced (Reviewed by Harrison, 2002) • No reports of XOR generation of NO in plants, although many tissues may be anaerobic to oxidoreductase by mammalian enzyme under allow such activity(XOR) low oxygen tensions

in vitro

Nitrite: NO- • Nitrite-reducing activity Tobacco (Stöhr

et al

., 2001) • A potentially important enzyme which needs further characterisationreductase resulted in NO production

Nonenzymatic • Nitrogen dioxide to NO (Cooney

et al

., 1994) • Requires low pH and a reductant such as ascorbatesources catalysed by carotenoids

• Nitrite to NO conversion Barley aleurone apoplast• NO

2

converted to NO (Bethke, P and Jones, R, pers. comm.)Rush, Lawn Grass, Ginkgo leaves(Nishimura

et al

., 1986)

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mammalian enzymes, several groups have provided evidencefor the existence of NOS in plants. Kuo et al. (1995) used amouse anti-brain NOS anti-body to show the presence of NOSin wheat germ. At the same time, others reported the presenceof immunoreactivity to anti-NOS antibodies in plant tissues.Using a rabbit anti-nNOS antibody, pea embryonic axes wereshown to contain a 105-kDa protein whilst analysis of wheatgerm revealed two bands: 58 kDa and 90 kDa (Sen & Cheema,1995). However, despite the now fairly prevalent immunolo-gical data for the presence of NOS in plants (discussed fur-ther below), caution should be exercised, as false positives canand do occur (Lo et al., 2000).

In 1996, Ninnemann and Maier showed, for the first time,the presence of NOS activity in higher plants. They used inhi-bition by the arginine analogues NG-nitro--arginine (L-NNA)and Nω-nitro--arginine methyl ester (-NAME), and theproduction of radiolabelled citrulline as evidence. Ribeiroet al. (1999) used a combination of antibody and activityassays to provide evidence for the presence of NOS in maize.Western blot analysis of the soluble fractions from roots andyoung leaves using anti-mouse iNOS and anti-rabbit nNOSrevealed a band at 166 kDa. In addition, the enzyme extractswere capable of converting 14C-arginine to 14C-citrulline.Together, these data suggest the existence of NOS in thisplant. Further work using the antibodies revealed that theNOS protein was located in the cytoplasm of the cells, buttranslocated to the nucleus, with translocation being depend-ent on the phase of cell growth (Ribeiro et al., 1999).

NOS activity was also reported in peroxisomes from pealeaves. Again using the arginine-citrulline assay, a calcium-dependent activity was found. The presence of NOS was fur-ther substantiated by the use of a raft of NOS inhibitors, ofwhich aminoguanidine was found to be the most effective.Interestingly, the NOS activity was also inhibited by the addi-tion of an anti-iNOS antibody (Barroso et al., 1999). Furtheranalysis with antibodies revealed a band on Western blots ofapproximately 130 kDa, and the immunoreactive protein waslocalised to peroxisomes and chloroplasts, but not mitochon-dria. Recent work using antibodies has shown that NOS andcatalase co-localise to perixosomes, the latter enzyme being acharacteristic marker for these organelles, and laser confocalimmunofluorescence has been used to visualise the location ofNOS. The presence of NO in peroxisomes was also furthersubstantiated by fluorometric analysis and electron paramag-netic resonance spectroscopy (EPR) using Fe-MGD (Corpaset al., 2002).

Other workers have reported NOS-like activity in plants inresponse to various stimuli. Bacterial challenge induced rapidNO generation in soybean suspension cultures (Delledonneet al., 1998), with the effect reduced by the NOS inhibitorL-NNA. Tobacco mosaic virus (TMV) infection of resistanttobacco also resulted in increased NO production that wasinhibited by a NOS inhibitor (Durner et al., 1998). NOSactivity was detected in roots and nodules of Lupinus albus,

and inhibited by the NOS inhibitor L-NMMA (NG-monomethyl--arginine; Cueto et al., 1996). In Arabidopsis,mechanical stress also induced NO production, and inhibi-tion by L-NMMA led Garcês et al. (2001) to conclude thatan inducible form of NOS is present. Foissner et al. (2000)showed the release of NO from tobacco using real-time imag-ing with confocal microscopy and the NO sensitive fluoro-phore diaminofluorescein diacetate (DAF-2DA; Kojimaet al., 1998). This activity was induced by a fungal elicitorfrom Phytophthora cryptogea, and also inhibited by the NOSinhibitor L-NMMA.

However, despite this growing evidence for the existence ofNOS in plants, other groups have reported conflicting data.For example, NOS inhibitors had no effect on NO synthesisin leaf extracts or intact tissues (Rockel et al., 2002), whileClarke et al. (2000) found no effects of NOS inhibitors on therelease of NO in Arabidopsis thaliana cells in response to bac-terial challenge. NOS inhibitors did not reduce nitrite-dependent NO generation in Chlamydomonas reinhardtii, nordid the addition of -arginine, the known substrate for NOS,induce activity (Sakihama et al., 2002). -arginine was simi-larly ineffective as a substrate for NO production in the greenalga Scenedesmus obliquus, and two known NOS inhibitorshad no effect (Mallick et al., 2000).

There is little evidence for the presence of genes encodingNOS in the Arabidopsis genome, suggesting that in this spe-cies at least, the presence of NOS is unlikely. However, it can-not be ruled out. Considering that NOS is a bi-domainenzyme, with a reductase domain very similar to that of P450reductase (an enzyme to which many of the antibodies usedto show the presence of NOS might be binding), it is possiblethat in plants all that is required for NO generation from sucha system would be a dedicated oxidase enzyme, being fed elec-trons from a common reductase. Such an oxidase could be rel-atively small, as long as it held a haem in the correct redoxmid-point potential. As haem chelation requires only two his-tidines, such an oxidase could be hard to identify using anti-mammalian antibodies, or mammalian-like gene sequences.

3.2 Nitrate reductase (NR)

It seems clear that even if NOS can be a source of NO inplants, alternate sources must also be present, with NR beingseen as the most likely candidate (Yamasaki et al., 1999;Yamasaki & Sakihama, 2000; see Table 1). In fact, over 20 yearsago, nitrogen oxides were reported in in vivo assays of soybeanleaves (Harper, 1981). Using nitrate reductase-deficientmutants of soybean, Dean & Harper (1986) found that suchplants did not evolve NO, unlike wild type plants, indicatingNR as a likely enzyme candidate for NO production. Theseworkers isolated and characterised the soybean NR activity,showing that it was NAD(P)H-dependent, had a pHoptimum of 6.75, and was cyanide sensitive (Dean & Harper,1988).

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Nitrate reductase is a key enzyme of nitrate assimilation inhigher plants (Pattanayak & Chatterjee, 1998; Lea, 1999),often catalysing the rate-limiting step. It uses NAD(P)H as anelectron source for the conversion of nitrate to nitrite (Lea,1999). NR also has the capacity to generate NO, an activitythat has been demonstrated in vitro (Yamasaki & Sakihama,2000) and in vivo (Rockel et al., 2002). The enzyme generatesNO from nitrite, again with NAD(P)H as an electron donor(Kaiser et al., 2002). NO is probably generated using MoCo(molybdenum cofactor) as the site of catalysis, as found inanother MoCo NO-producing enzyme, xanthine oxidore-ductase (Harrison, 2002). However, in vitro, the NO gener-ating capacity of NR could only account for a small part(< 1%) of the total NR activity extracted (Rockel et al., 2002).The Km for nitrite has been found to be approx. 100 µ, aconcentration higher than the endogenous nitrite concentra-tion estimated in illuminated spinach leaves (10 µ), and theactivity was competitively inhibited by nitrate (Ki ∼ 50 µ).For example, the infusion of nitrate through leaf petiolesdecreased NO generation. Therefore, the rate of NR-generatedNO in vivo will be dependent on the intracellular concen-tration of both these compounds, as well as the enzymaticturnover rate of the enzyme itself (Rockel et al., 2002). Intra-cellular nitrite has been estimated to be between 10 µ and4.8 m in spinach (Rockel et al., 2002), concentrations thatcorrelate with the findings of others (Siddiqi et al., 1992),while nitrate concentrations have be reported to be in the mil-limolar range (Miller & Smith, 1996). Using nitrite reductase(NiR) antisense NiR tobacco, Morot-Gaudry et al. (2002)detected elevated endogenous nitrite levels, with a concomi-tant rise in NO release, indicating a link between nitrite andNO. It should be noted that like other NO-generatingenzymes such as NOS, NR can also produce peroxynitrite(Yamasaki & Sakihama, 2000).

In higher plants NR is usually found as homodimer, withsubunits of 100–115 kDa, depending on the species studied,although in some species it is tetrameric. The spinach NR has926 amino acids, while others may be a little smaller: the beanNR being 881 amino acids (Hoff et al., 1994). Its catalytic actionrequires electron transfer, a process that involves three prostheticgroups, FAD, haem and MoCo. Kinetic analysis revealed thatno single step in the electron transfer was rate-limiting (Skipperet al., 2001). Interestingly, the topology of the protein revealsthree structural domains, one for each prosthetic group, sep-arated by hinge regions that are susceptible to proteolyticcleavage (Fig. 3). Towards the N-terminus is the MoCodomain, similar to mammalian sulphite oxidase, with the haembinding region, involving histidine residues and showing sim-ilarities to cytochrome b5, lying in a central domain. The FADbinding site is found in the third domain, towards the C-terminal end of the polypeptide, and this domain is similar tocytochrome b5 reductase (Campbell, 1996). Thus, like NOS,NR appears to be constructed of domains which when cleavedfrom the holoenzyme can have autonomous activity.

Expression of the NR genes is light-dependent, following adiurnal pattern, as well as being nitrate-inducible (Hoff et al.,1994). Light appears not only to induce the transcription ofNR genes, but also influence the production of the proteinitself, either through control of translation, or by influencingthe stability of the protein once synthesised (Vincentz &Caboche, 1991).

Control of NR activity is via covalent modification, involvingphosphorylation and dephosphorylation. NR is rapidly inac-tivated by phosphorylation following a light to dark transi-tion, the site of phosphorylation being serine-543 (in spinach),an amino acid conserved in NR sequences of higher plants(Rouze & Caboche, 1992; Hoff et al., 1994; Bachmann et al.,1996a). Phosphorylation may be Ca2+ − dependent (Bachmannet al., 1996a,b; Huber et al., 1996). The NR phosphoproteinis recognised by a NR inhibitory protein (NIP), a member ofthe 14-3-3 family of controlling polypeptides (Bachmannet al., 1996b; Moorhead et al., 1996; Kaiser & Huber, 2001).Binding of NIP and NR inactivates NR. The rates of degra-dation of NR are also thought to be dependent on its phos-phorylation state and association with the 14-3-3 protein.

Experimentally, the activity of NR has been modulated bythe addition of tungstate. Certainly, pretreatment with tung-state reduces the subsequent NR activity in cells. Tungstateserves as a molybdenum analogue, and the reduction in NRactivity in plants is caused by the synthesis of an inactive tung-stoprotein (Notton & Hewitt, 1971a). In fact, mRNA levelsencoding NR, and levels of NR protein, are increased ontungstate treatment, although activity is diminished (Denget al., 1989). No direct inhibition of NR by tungstate hasbeen reported. However, we have found that abscisic acid(ABA)-induced NO synthesis in Arabidopsis guard cells, andconcomitant ABA-induced stomatal closure, are both inhib-ited by tungstate (R. Desikan et al. unpublished). Moreover,these ABA effects are not inhibited by the NOS inhibitorL-NAME. These effects are in contrast to those observed forpea, suggesting the data are physiologically relevant. In Arabi-dopsis, tungstate did not inhibit stomatal closure induced byNO, H2O2 or darkness, indicating that its effects were specificfor NO synthesis. Clearly, it will be important to assess theeffects of tungstate on NR activity, both in vitro and in vivo.NR can also be inhibited by cyanide (Notton & Hewitt,1971b) or azide (Yamasaki & Sakihama, 2000), but thesehave limited experimental value as they are known to inhibitmany other enzymes, for example cytochrome oxidase.

Undoubtedly, the identification of plants lacking in NRactivity, or at least with severely depleted activity, will aidgreatly in the identification of the role of NR in plants – forexample, the nia1, nia2 Arabidopsis mutant, in which bothNR genes are mutated (Wilkinson & Crawford, 1993), aswell as those of other species such as soybean (Dean & Harper,1986). NR-deficient mutants of Arabidopsis were instrumen-tal in our work to investigate the role of NR in ABA-inducedNO generation (see section 4 below). Further work will no

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doubt show that NR, and its generation of NO, are involvedin many more physiological responses in plants.

NR was also found to be the source of NO in the green algaChlamydomonas reinhardtii (Sakihama et al., 2002). Using aNO-specific electrode and the fluorescence probe DAF-2DA,nitrite was shown to induce NO generation in the dark, aresponse that was suppressed in light. Furthermore, in a NR-lacking mutant, cc-2929, the response was annulled, provid-ing good evidence that NR was the source of NO.

As might be expected, NR activity in spinach leaves wasreduced by the addition of phosphatase inhibitors (Rockelet al., 2002). NR activity was also increased by the addition ofuncouplers, while inactive NR was activated by rises in5′AMP (Kaiser et al., 1999). Along with anoxia, both 5′AMPand uncouplers led to a rise in nitrite concentration in thecells, and a rise in NO generation (Rockel et al., 2002). Simi-lar observations, showing the modulation of NR activity by5′AMP, have also been made in cucumber (de la Haba et al.,2001).

3.3 Other enzymatic sources of NO

Other enzymes can also generate NO (Table 1). For example,Stöhr et al. (2001) demonstrated nitrite-reducing activity intobacco roots, which resulted in the generation of NO. Theplasma membrane-enzyme was designated as nitrite: NO-reductase (Ni-NOR). It was insensitive to cyanide and to anti-NR IgG, and gel-filtration showed that it had an apparentmass of 310 kDa, much larger than the 200 kDa estimatedfor NR. Such an enzyme could be very important, especiallyif NO is acting as an intercellular signal, as it is well placed torelease NO from the cell. Clearly, there is a need tocharacterise this enzyme and to determine its species andtissue distribution.

Xanthine oxidoreductase (XOR: otherwise referred to asxanthine oxidase (XO) or xanthine dehydrogenase (XDH)), isalso an enzyme recently shown to produce NO (Harrison,2002). Like NR, this is also a redox enzyme with a molybde-num cofactor. NO-generating activity is increased at low oxy-gen tensions, in catalysis which, like NR, sees the conversionof inorganic nitrite to NO (Millar et al., 1997; Godber et al.,2000a). If oxygen is present, superoxide is also produced.Therefore, at near anaerobic oxygen tensions, a competitionfor electrons is established, with the enzyme producing bothsuperoxide and NO. The superoxide can subsequently reactwith NO to form peroxynitrite (Godber et al., 2000b), andthis has been suggested as an antibacterial mechanism inmammals (Hancock et al., 2002). Therefore, this enzyme hasthe capacity to produce two signals – either superoxide (whichwill dismute to H2O2) and ROS signalling if oxygen tensionsare high, or NO signalling if oxygen tensions drop. As someplant tissues such as roots can become temporarily anaerobic,this enzyme offers an exciting signalling scenario, where sig-nals can be modulated by the availability of oxygen. Xanthine

oxidase activity has been found in plant peroxisomes (Corpaset al., 2001). The role of this enzyme in plant signalling needsto be clearly established, and little work in this area has beencarried out to date.

3.4 Nonenzymatic sources of NO

NO can also be generated from nonenzymatic sourcesunder the correct conditions. If the environment is par-ticularly acid or reducing, then chemical reduction ofnitrite will yield NO, with the chemistry proceeding througha step involving nitrous acid, while ascorbate can react withnitrous acid to yield dehydroascorbic acid and nitric oxide(Weitzberg & Lundberg, 1998). Such reactions can occur inplant tissues. For example, it has been shown recently thatbarley aleurone cells can generate a sufficiently acidicapoplastic environment to support nitrite to NO conversionusing ascorbate as a reductant (P. Bethke & R. Jones pers.comm.).

Light-mediated conversion of nitrogen dioxide to nitricoxide can be catalysed by carotenoids (Cooney et al., 1994),although this requires an acid pH and will only occur inselected compartments in the cells. NO2 was also reported tobe absorbed by rush, lawn grass and ginkgo leaves, andreleased as NO. The potential reductant was fractionated andidentified as a polysaccharide (Nishimura et al., 1986).

4. NO biology

4.1 Experimental approaches

Application of NO to plants and induction of subsequentresponses does not, of course, by itself prove that endogenousNO mediates the particular developmental or physiologicalprocess under study. Rather, as with plant hormones, variouscriteria should be met in order to provide convincing evidenceof a role for endogenous NO. These include induction of theprocess following NO application; inhibition correlated withinhibition of NO synthesis via chemical or genetic means;inhibition of the response following removal of the NO viaNO-scavengers; and correlation of NO synthesis/concen-tration with the particular biological process. Typically, NO isapplied to plants (and most organisms) via an NO-donor – thatis, a molecule that will generate NO, sometimes after passageinto cells. This approach is technically simple (compared tothe application of NO gas which has only rarely been used).A range of concentrations can be used and, as NO release canoften be measured, it is possible to investigate NO dose–responses. Various NO donors have been employed, with theassumption that they release NO. Probably the most commonlyused is sodium nitroprusside (SNP), a compound that in factis likely to generate NO+ (Stamler et al., 1992). Other NOdonors include S-nitrosoglutathione (GSNO), a compound thatdoes release NO but that may have other effects (Hogg, 2000),

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SNAP (S-nitroso-N-acetylpenicillamine; Durner et al., 1998),RBS (Roussin’s Black Salts; Clarke et al., 2000) and NOR-3((+/–)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide;Huang et al., 2002). In order to be confident that the effectsof NO donors are truly due to NO release, and not due to thechemicals per se, it is useful to use more than one donor. It isalso useful to include suitable controls – for example theresidual chemical after cleavage and release of NO, and toshow that the effects of the donor can be negated byapplication of an NO scavenger. The NO scavengers PTIO(2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide) andcPTIO (carboxyPTIO) are commonly used. PTIO is sup-posedly specific for NO but cPTIO has been reported toinhibit NOS; in fact, any pharmacological approach has tobe edged with caution, as most biologically active moleculesare likely to have side-effects. Several NOS inhibitors such asL-NAME widely used in mammalian studies have also beenused with plants (see section 3 above). As no NOS has yetbeen cloned from plants, there are clearly no NOS– mutantsyet available. However, NR– mutants are available, and havebeen used to provide evidence both for and against the NO-generating involvement of NR in biological processes (Garcêset al., 2001; Desikan et al., 2002).

4.2 Demonstration and quantification of NO

Ideally, NO should be quantified accurately and reliably at itscellular site of action. This means that any technique shouldmeasure NO and not other molecules, and not be prone tointerference. In fact, NO quantification is problematic forvarious reasons. In a similar way to ethylene measurements,NO emission from plants has been estimated by gaschromatography and mass spectrometry (Dean & Harper,1986; Magalhaes et al., 2000; P. Bethke & R. Jones, pers.comm.), chemiluminescence (Wildt et al., 1997) and usinglaser photo-acoustic spectroscopy (Leshem & Pinchasov,2000). This latter technique is very sensitive and specific andis being used to record reliable changes in NO emissions fromplants subject to various treatments, e.g. pathogen infections(L. Mur, pers. comm.). It is assumed that rates of NOemission reflect global rates of NO synthesis and reactionsalthough it is difficult to know if they actually reflect NOconcentrations in target cells. NO can be assayed in solutionusing an NO-electrode, that measures the steady-stateconcentration attained by release from the source of NO, bethat an NO-donor or cells releasing NO. NO can also bemeasured by reaction with molecules such as haemoglobin,that reacts with NO and displays a spectral shift that can bedetected and used to quantify the accumulation of NO(Murphy & Noack, 1994). Haemoglobin can also react withother molecules such as ROS that may be present with NO.NO-sensitive fluors have been developed, such as DAF-2DA(Kojima et al., 1998). DAF-2DA is a cell-permeable moleculethat does not fluoresce until it reacts with NO, and can thus

be used to monitor relative intracellular NO content usingfluorescence and confocal microscopy. Although DAF-2DAdoes not react with ROS (Foissner et al., 2000) there arereports of nonspecific (i.e. non-NO) fluorescence (Beligniet al., 2002). The non-NO-reactive analogue 4-AF is a usefulcontrol (Desikan et al., 2002; Mata & Lamattina, 2002).Reaction with calcium has also been reported to increase thesensitivity of DAF-2DA to NO (Broillet et al., 2001), animportant point as calcium elevations represent a commonsignalling response in cells. DAF-2DA fluorescence canclearly report changes in NO within plant cells, but it isdifficult to equate changes in fluorescence with the actualconcentration of NO in solution. In epidermal and guardcells, DAF-2DA fluoresence is commonly at its most intensein chloroplasts (Foissner et al., 2000; Desikan et al., 2002;Neill et al., 2002a) but this might simply reflect DAF-2DAaccumulation in these organelles. EPR spectroscopy can alsobe used to quantify NO within tissues (Pagnussat et al.,2002). NO will react with ‘spin-trap’ probes to form a stableadduct with characteristic EPR spectral properties that canthen be monitored.

Given the technical and conceptual problems, it is notsurprising that there are relatively few data available on quan-tification and detection of NO. Leshem & Haramaty (1996)reported up to 160 n/g f. wt of NO from wilted pealeaves. In Arabidopsis, Magalhaes et al. (2000) observed500 nl/g f. wt h−1 of NO being emitted from wild type plants.In Arabidopsis cells, up to 180 nmol/g f. wt h−1 of NO releasehas been detected following pathogen challenge (Clarke et al.,2000), and an endogenous NO content of 55 nmol/g f. wtwas measured in auxin-treated cucumber roots (Pagnussatet al., 2002).

4.3 NO and plant growth and development

It has been known for some time that plants emit NO undernormal growing conditions, and that NO can accumulate inthe atmosphere from various sources, including industrialpollution (Wildt et al., 1997 and references therein).Detrimental effects of NO on photosynthesis were reportedmany years ago and subsequently the effects of NO on plantgrowth were found to be concentration dependent (Anderson& Mansfield, 1979). High levels (40–80 pphm) inhibitedtomato growth, whereas low levels (0–20 pphm) enhancedgrowth, findings repeated for lettuce (Hufton et al., 1996)and pea (Leshem & Haramaty, 1996). Recent data fromTakahashi & Yamasaki (2002) show that NO can reversiblysuppress electron transport and ATP synthesis in chloroplasts.As nitrite can be a source of NO, it was suggested that, underconditions where nitrite reduction by nitrite reductase islimited, NR-produced NO could inhibit photosynthesis. Inaccord with this suggestion, antisense-nitrite reductase tobaccoplants have been found to accumulate increased NO andexhibit reduced growth (Morot-Gaudry et al., 2002).

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The effects of NO on hypocotyl and internode elongationhave also been reported. NO donors inhibited hypocotylgrowth and stimulated de-etiolation and an increase in chlo-rophyll in potato, lettuce and Arabidopsis (Beligni & Lamattina,2000). NO also increased chlorophyll content in pea leaves,particularly in guard cells (Leshem et al., 1997), and retardedchlorophyll loss in Phytophthora infestans-infected potatoleaves (Laxalt et al., 1997). The positive effects of NO onchlorophyll retention may reflect NO effects on iron avail-ability. Graziano et al. (2002) have shown that iron availabil-ity is improved in the presence of NO. Iron deficiency resultsin chlorosis caused by reduced chloroplast development, andNO treatment of wild type maize inhibited such chlorosis.Furthermore, iron deficiency in yellow stripe mutants wasreversed by NO application (Graziano et al., 2002).

Some reports indicate that NO may have anti-senescenceproperties. Leshem & Haramaty (1996) found that applicationof an NO donor to pea leaves under senescence-promotingconditions decreased generation of ethylene, an endogen-ous driver of senescence, that was subsequently shown toresult from an inhibition of ethylene biosynthesis (Leshem,2001). On the other hand, Magalhaes et al. (2000) observedthat exposure of Arabidopsis plants to NO gas increasedethylene levels, and that inhibition of NO synthesis didnot affect ethylene accumulation. A potential role for NOin delaying flower senescence is indicated by the increasedlongevity of several varieties of cut flowers induced by appli-cation of NO donors (Leshem, 2001).

Fruit ripening is another senescence-related process pro-moted by ethylene that can be retarded by NO. Increased ethy-lene production during ripening of fruits such as banana andstrawberries coincides with reduced NO emission (Leshem &Pinchasov, 2000; Leshem, 2001). In addition, application ofNO to vegetables and fruits also delayed senescence andextended their postharvest life.

NO can stimulate seed germination in various plant spe-cies. Application of NO donors broke dark-imposed seed dor-mancy in lettuce, that was reversed in the presence of the NOscavenger PTIO (Beligni & Lamattina, 2000). Such effects ofNO might also explain the germination of dormant seeds ofCalifornia chaparral plants induced by smoke containingnitrogen oxides (Keeley & Fotheringham, 1997). Giba et al.(1998) used various NO donors and the appropriate inactivecompounds, to demonstrate that phytochrome-controlledgermination of Empress tree seeds was mediated by NO. Atlow pH (2.5 and 3), nitrite promoted seed germination, andacidic conditions minus nitrite did not (Giba et al., 1998).NO was generated from nitrite under low pH conditions(Giba et al., 1998), and nitrite-induced NO synthesis via anonenzymatic route can occur in plants (see section 3.4above). Thus, some early reports on the promotion of seedgermination by nitrite might be explained by NO generation.For example, Hendricks & Taylorson (1974) and Cohn et al.(1983) reported that nitrite promoted germination of seeds

from various plant species. These effects of nitrite (and thusNO) on seed germination are interesting, and suggest that thelevel of soil nitrite is one factor determining seed germination.

New roles for NO in plant growth and development arelikely. For example, a recent report (Pagnussat et al., 2002) hasprovided exciting data on NO and root development. NO(applied via the NO donors SNP and SNAP) induced adven-titious root development in cucumber. Auxin-induced rootgrowth and formation of lateral roots was also blocked by theNO scavenger cPTIO (Pagnussat et al., 2002). A previousreport had suggested a role for NO in root elongation inmaize, although in this case the effects of auxin were notreversed by a NO scavenger (Gouvea et al., 1997).

4.4 NO and hormones

It may well be that in some cases, NO mediates the biologicaleffects of primary signalling molecules such as hormones, asituation analogous to that being revealed for H2O2 (Neillet al., 2002b,c). For example, hormones may be transportedfrom one location to another and therein induce NOsynthesis, or hormone sensitivity may alter developmentallyor in response to an environmental stimulus, such that cellsacquire the competence to respond to hormones by way ofNO synthesis. In either case, NO synthesis might be restrictedto specific target cells. So far, cytokinin has been shown toinduce NO synthesis in tobacco, parsley and Arabidopsis cellcultures (Tun et al., 2001). Some cytokinin effects can bemimicked by NO – NO donors induced betalaine accu-mulation in Amaranthus seedlings, and cytokinin-inducedbetalaine accumulation was inhibited by a NOS inhibitor(Scherer & Holk, 2000), suggesting that NO might mediatesome cytokinin effects. A novel role for cytokinins in theinduction of programmed cell death (PCD) has recently beenproposed (Carimi et al., 2002). Given that NO induces PCD(see section 4.7 below), it is possible that NO mediates thiscytokinin-induced process. Cytokinin-response mutants suchas cki1 (Kakimoto, 1996) will be useful tools here.

The recent discovery that NO mediates ABA-induced sto-matal closure represents a significant development in NOresearch. Stomatal movements are effected by osmotic fluxesof water across the tonoplast and plasma membrane, suchfluxes being driven by movements of K+ and Cl– ions throughspecific channels that are activated and deactivated inresponse to various stimuli such as ABA (Schroeder et al.,2001). Guard cell signalling is highly complex, but mostsignals elicit changes in cytosolic calcium concentrations,often in an oscillating manner (Schroeder et al., 2001).Calcium increases are induced by signalling molecules such asinositol trisphosophate (IP3) and hexakisphosphate (IP6),sphingosine-1-phosphate (S1P), H2O2 and cyclic adenosine5′-diphosphoribose (cADPR) (Hetherington, 2001). Other sig-nal transduction mechanisms in guard cells include altera-tions in pH, cytoskeletal arrangement, gene expression and

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membrane trafficking (Hetherington, 2001; Schroeder et al.,2001). Recent work has identified novel facets of ABA signaltransduction in guard cells, such as mediation of calciumrelease by H2O2 and S1P (Hetherington, 2001; Schroederet al., 2001), the involvement of G-proteins (Wang et al.,2001) and the role of protein phosphorylation and RNA-binding proteins (Li et al., 2002).

In our laboratory, we have shown recently that ABAinduces rapid NO synthesis in guard cells (and other epidermalcells) of pea (Neill et al., 2002a). This finding has since beenextended to Vicia faba (Garcia-Mata & Lamattina, 2002) andArabidopsis (Desikan et al., 2002). ABA did not induce NOsynthesis in Arabidopsis suspension cultures (Tun et al., 2001),indicating tissue specificity. In pea, ABA-induced NO synthe-sis in guard cells was required for stomatal closure, as removalof NO with the scavenger PTIO substantially inhibited ABA-induced closure. Pharmacological evidence indicated NOS asa source of NO, as ABA-induced stomatal closure and NOsynthesis were both inhibited by L-NAME (Neill et al.,2002a), but not by tungstate, a potential NR inhibitor(R. Desikan et al., unpublished). ABA-induced NO synthesisis also required for ABA-induced stomatal closure in Arabi-dopsis (Desikan et al., 2002; Fig. 4). Here, however, the sourceof NO appears to be NR, not NOS. Tungstate strongly inhib-its ABA-induced NO synthesis and stomatal closure (see sec-tion 3.2 above) but L-NAME has no effect (Desikan et al.,2002). Consistent with this finding, nitrite induces NO accu-

mulation in guard cells and stomatal closure, both eventsbeing negated by a NO scavenger (Desikan et al., 2002 andFig. 4). Moreover, guard cells of the NR-deficient Arabidopsisnia1, nia2 mutant (Wilkinson & Crawford, 1993) do notsynthesise NO nor do they close in response to nitrite or ABA(Desikan et al., 2002). It seems possible, then, that there existspecies differences in NO synthesis, at least in terms of guardcell responses to ABA.

NO synthesis has recently been found to be induced byauxin in cucumber roots (Pagnussat et al., 2002). NO wasrequired for root growth and the formation of lateral roots,but the source of NO has not yet been determined.

4.5 NO and abiotic stress

It is well known that various abiotic stresses such as drought,low and high temperatures, UV and ozone exposure inducethe generation of ROS (Neill et al., 2002b; Vranova et al.,2002). ROS initiate several oxidatively destructive processes,but also trigger various signalling pathways (Neill et al.,2002b; Vranova et al., 2002). Thus, maintenance of appro-priate ROS levels might represent a survival response. In fact,NO interacts with ROS in various ways and might serve anantioxidant function during various stresses (Beligni & Lamat-tina, 1999). Modulation by NO of superoxide formation(Caro & Puntarulo, 1998) and inhibition of lipid peroxidation(Boveris et al., 2000) also illustrate its potential antioxidant

Fig. 4 Nitric oxide (NO) generated in guard cells in response to abscisic acid (ABA; 50 µM) leads to stomatal closure. (a) Arabidopsis guard cells treated either without (top left) or with (top right) ABA, in the presence of the NO scavenger PTIO (bottom right), or treated with nitrite (bottom left). Guard cells visualised using confocal microscopy and a NO-sensitive fluor DAF-2DA. (b) ABA-induced stomatal closure in epidermal peels. (c) Time course of ABA-induced NO synthesis in guard cells.

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role. On the other hand, excess NO can result in nitrosativestress (see section 5.5 below), so a favourable balance ofROS/NO is important.

Drought stress is a major environmental constraint on cropproductivity and performance, and understanding the cellularprocesses that ameliorate the consequences of drought stressand conserve water are clearly important. ABA is synthesisedfollowing turgor loss and stimulates guard cell NO synthesis,but the effects of dehydration on NO generation have not yetbeen resolved. Leshem & Haramaty (1996) reported thatwilting increased NO emission from pea plants. However,working with Arabidopsis, Magalhaes et al. (2000) found theopposite. This might represent species differences, but it willbe important to determine the effects of water stresses at arange of reduced water potentials and over varying time peri-ods. Nevertheless, application of NO donors does reduce sto-matal apertures and thereby reduce transpiration in severalspecies (Mata & Lamattina, 2001). It is likely that NO doesnot act alone, but interacts with other signalling moleculessuch as H2O2, to effect stomatal closure. Preliminary dataindicate that both H2O2 and NO are required for full sto-matal closure in Arabidopsis (R. Desikan et al., unpublished).There is also some evidence that ROS and NO interact toinduce ABA biosynthesis. In response to drought stress, anincrease in NOS-like activity was observed in wheat seedlings,and ABA accumulation was inhibited by NOS inhibitors(Zhao et al., 2001). ABA-induced NADPH oxidase activityduring drought stress, leading to increased ROS levels, hasalso been reported for maize ( Jiang & Zhang, 2002), indicat-ing a close interplay between ABA, ROS and NO levels. Theuse of ABA, ROS and NO mutants should help to elucidatethese complex interactions.

NO responses to other stresses such as heat and chillinghave also been noted. Short-term heat stress caused anincrease in NO production in alfalfa (Leshem, 2001). Appli-cation of NO mediates chilling resistance in tomato, wheatand corn (Lamattina et al., 2001). It is possible that this effectreflects the antioxidant properties of NO, via suppression ofthe high levels of ROS that accumulate following exposure tochilling or heat stress (Neill et al., 2002b).

Using NO donors and a NOS inhibitor, Mackerness et al.(2001) have shown that UV-B-induced expression of chal-cone synthase (CHS ) occurs via a NOS-like enzyme.Although this is the first demonstration of a role for NOS andNO in UV-B responses, it remains to be seen whether UV-Btreatment actually causes an increase in NO synthesis, and ifNO is also involved in other responses to UV-B radiation. Inrelation to this, treatment of potato tubers with NO donorsbefore UV irradiation was found to result in 50% morehealthy leaves compared to plants not pre-treated with NO(Lamattina et al., 2001).

Another atmospheric pollutant that might interact withNO is ozone. Ozone treatment of Arabidopsis plants inducedNOS activity that preceded accumulation of salicylic acid

(SA) and cell death (Rao & Davis, 2001). In tobacco, NO wasfound to induce SA synthesis (Durner et al., 1998). Moreover,NO treatment has been shown to increase the levels of ozone-induced ethylene production and leaf injury (Rao & Davis,2002).

Wounding is a common consequence of pathogen chal-lenge of plants, during which the generation and increasedaccumulation of NO and H2O2 are frequently observed(Delledonne et al., 1998). Orozco-Cardenas & Ryan (2002)demonstrated that although wounding per se does not inducethe generation of NO, treatment with NO donors inhibitedH2O2 generation following wounding, as well as the expres-sion of specific wound-induced genes. This suggests that NOproduced during pathogenesis might inhibit H2O2 synthesisand the activation of specific wound-induced signallingpathways.

4.6 NO and biotic interactions

The publication of two papers in 1998 describing the role ofNO in plant defence signalling (Delledonne et al., 1998;Durner et al., 1998) led to a big increase in NO research. Boththese papers demonstrated a key signalling role for NO duringthe induction of the Hypersensitive Response (HR). HR is adefence process activated in plants in response to pathogenattack. Associated with the HR is an oxidative burst, in whichthere is greatly increased ROS generation, PCD, and theactivation of signalling pathways driving the expression ofvarious defence-related genes. HR results in localised plantcell death, which in turn limits nutrient availability and thusgrowth and spread of the invading pathogen. Treatment ofsoybean cultures with avirulent (HR-inducing) but notvirulent (HR-noninducing) Pseudomonas syringae pv glycinea,induced rapid NO synthesis with kinetics similar to H2O2generation (Delledonne et al., 1998). Moreover, NO donorsinduced cell death (but only during rapid mechanicalagitation of the cells, indicating an interaction between NOand mechanically induced H2O2) as well as expression ofphenylalanine ammonia-lyase (PAL) and CHS genes.Furthermore, bacterially induced cell death and PAL geneexpression were blocked by NOS inhibitors, and constitutiveNOS activity was identified in cytosolic fractions of soybean.Induction of HR in Arabidopsis leaves by P. syringae pvmaculicola was also reduced by NOS inhibitors. Together,these data indicate that pathogen-induced NO produced viaNOS, interacts with H2O2 to mediate the HR.

Durner et al. (1998) also provided compelling evidencefor the role of NO during plant defence responses. Infectionof tobacco plants with HR-inducing varieties of tobaccomosaic virus (TMV) induced NOS activity that was inhibitedby NOS inhibitors. NO also induced the synthesis of SA andexpression of the defence-related gene PR-1. SA is a defencesignalling molecule involved in the development of systemicacquired resistance (SAR; Draper, 1997). Thus, the data of

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Durner et al. (1998) suggested a role for NO in SAR. Giventhat SA treatment leads to enhanced NO production (Klepper,1991) a complex signalling relationship between H2O2, NOand SA during HR and SAR is likely (Van Camp et al., 1998;Song & Goodman, 2001).

Accumulation of phytoalexins is another phenomenonassociated with the HR during pathogen challenge. Treatmentof potato tubers with a NO donor stimulated an increase inthe accumulation of the phytoalexin rishitin (Noritake et al.,1996). This NO-mediated effect was inhibited by treatmentwith the NO scavenger PTIO. However, elicitor-induced ris-hitin accumulation was not affected by PTIO, suggesting thatperhaps in this case, endogenous NO is not involved in thedefence response. More recently though, a role for endog-enous NO in phytoalexin biosynthesis was described. NOtreatment of soybean cotyledons triggered the biosynthesis ofphytoalexins (Modolo et al., 2002). Furthermore, elicitor-induced phytoalexin formation was inhibited by NOS inhi-bitors, implying NOS as a source of NO in this pathway. NOregulation of phenylpropanoid metabolism was reported byEnkhardt & Pommer (2000). In maize shoots, NO appearedto bind to the haem group of cytochrome P450, and thusaffect the activity of cinnamic acid hydroxylase, a cytochromeP450-dependent monooxygenase involved in phenylpropa-noid biosynthesis.

A role for NO during symbiosis has been suggested inbacteria–legume interactions. NO was detected in soybeannodules using EPR spectroscopy (Mathieu et al., 1998), and,more recently, using confocal microscopy in alfalfa nodules(Herouart et al., 2002). NOS immunoreactivity has beendemonstrated in Lupinus nodules (Cueto et al., 1996). How-ever, a clear function for NO during symbiosis has not yetbeen established. There have been some reports that NO actsas a negative regulator of nitrogen fixation due to its interac-tion with leghaemoglobin (Herouart et al., 2002). Other dataindicate that modulation of NO levels results in alteration ofnodule numbers (Herouart et al., 2002).

4.7 NO and PCD

PCD is a genetically determined, metabolically directedcellular process resulting in cell suicide – cells die because ofactivation of intrinsic signalling and execution processes,rather than by necrosis induced by damage of various sorts.PCD during the HR has been well studied. In our labora-tory, research has focussed on establishing a role for NO dur-ing pathogen-induced PCD in Arabidopsis cell cultures.Challenging Arabidopsis cells with avirulent but not virulentP. syringae pv maculicola induced NO synthesis, correlatedwith the generation of H2O2 (Clarke et al., 2000). NO-induced cell death also possessed the characteristics of PCD,such as chromatin condensation, requirement for geneexpression, and activation of a caspase-like cascade. Althoughthere is no molecular evidence for caspases in plants, a similar

role may be played by metacaspases (The ArabidopsisGenome Initiative, 2000). However, recent work has shownthat overexpression of a cysteine protease in Arabidopsis cellsresulted in an inhibition of pathogen- and NO-induced celldeath (M. Delledonne, pers. comm.), suggesting thatdifferences in NO-mediated signalling pathways leading toPCD are likely to occur in different systems.

Delledonne et al. (2001) have shown that the interactionbetween NO and ROS can determine whether or not PCD isthe outcome. NO by itself does not induce PCD in soybeancell cultures, but it may be that the NO: superoxide ratiodetermines PCD. If superoxide levels are greater than those ofNO, then NO reacts with superoxide to form peroxynitrite,which does not result in PCD. However, if more NO thansuperoxide occurs, then NO reacts with H2O2 (arising fromdismutation of superoxide) to induce cell death. A correlationbetween H2O2, NO and antioxidant levels has also been dem-onstrated recently by de Pinto et al. (2002). In tobacco BY-2cells, neither NO nor H2O2 alone at low concentrations hadany effect on PCD or on the activity of PAL. However, treat-ment with both H2O2 and NO together induced a substantialincrease in cell death with characteristics of PCD, as well asPAL activity. Moreover, this treatment also caused an increasein the activities of enzymes reducing ascorbate and glutath-ione (De Pinto et al., 2002), implying that both H2O2 andNO regulate cellular antioxidant levels to effect PCD, at leastin some systems.

Beligni et al. (2002) have provided data indicating an anti-oxidant role for NO acting during developmental PCDinduced by hormones. In barley aleurone layers, GA-inducedPCD was delayed in the presence of NO, which correlatedwith a delayed loss of activity of the antioxidants catalase andsuperoxide dismutase. However, NO did not inhibit GA-induced alpha-amylase expression and activity, suggestingthat NO does not have a general effect on cellular metabo-lism, but acts as a specific endogenous modulator of PCD(Beligni et al., 2002).

PCD occurring as a result of mechanical stress may alsoinvolve NO. In Kalanchoe daigremontiana, centrifugation ofleaves and callus induced NO generation and subsequentDNA fragmentation and cell death (Pedroso et al., 2000).Decreased NO synthesis and PCD in the presence of a NOSinhibitor suggested the involvement of a NOS-like enzymein this species. Further work by Garcês et al. (2001) showedthat mechanical stress of Arabidopsis tissues also induced NOgeneration via a NOS-like enzyme. Thus in Arabidopsis atleast, NO can be generated via different mechanisms underdifferent situations, as is becoming evident for H2O2 (Neillet al., 2002c)

PCD is correlated with altered mitochondrial function anda role for NO is emerging here. In mammalian cells, short-term exposure to NO inhibits mitochondrial respiration viareversible inhibition of complex IV. Prolonged NO exposureresults in a gradual and persistent inhibition of complex I,

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concomitant with a reduction in intracellular glutathioneconcentrations, ultimately leading to cell death (Clementiet al., 1998). In plant cells, NO inhibition of ATP synthesisin mitochondria via inhibition of cytochrome oxidase activityhas been demonstrated by Yamasaki et al. (2001). Alteredmitochondrial activity stimulates PCD in plant cells. NO-induced PCD in Arabidopsis cells occurred via inhibition ofrespiration and the release of mitochondrial cytochrome c(Zottini et al., 2002). Saviani et al. (2002) showed thattreatment of Citrus cultures with NO induced cell death bear-ing the characteristic hallmarks of PCD. Cyclosporin A, aninhibitor of mitochondrial permeability transition pore(PTP) formation, inhibited NO-induced PCD as well asmitochondrial membrane potential (Saviani et al., 2002).Thus, PTP formation is one of the molecular targets of NOto activate the PCD process in plants.

Therefore, it seems that NO-induced altered mitochon-drial function has a harmful effect on the plant cell. However,mitochondrial respiratory shut-down can be compensated byan alternative pathway, using alternative oxidase (AOX),found in plants, fungi and protozoa. Millar & Day (1997)observed that although NO inhibited mitochondrial respira-tion, electron flow via AOX was not affected. Yamasaki et al.(2001) have since shown that AOX functions to avoid exces-sive ROS generation, unlike the situation in mammalian cells,where inhibition of cytochrome oxidase leads to increasedROS generation. More recently, Huang et al. (2002) foundthat exposure to NO induced the expression of variousgenes, one of which included AOX. Inhibition of the AOXpathway increased NO sensitivity and cell death, suggesting a‘shunting’ of the respiratory pathway via AOX. IncreasedAOX activity has also been observed during pathogenchallenge of Arabidopsis and tobacco (Simons et al., 1999).

5. NO signal transduction

Although there are an ever-increasing number of NOresponses in plants, we still know relatively little of the signaltransduction processes by which NO interaction with cellsresults in altered cellular activities. It seems unlikely thatspecific receptors exist for NO, as NO is such a simple, smalland diffusible molecule. However, cells undoubtedly do senseNO, as various cellular activities are modulated in itspresence. Given the ability of NO to react with a range oftarget molecules it may be that there are in fact several cellular‘NO sensors’. It is important to reiterate that NO can exist inseveral different redox forms, that each might activate specificcellular events, and that various NO donors can generatedifferent NO species. Consequently, it would be useful todetermine which NO species are present, and when. The ratioof NO·, NO+ and NO– and derivatives thereof may wellchange depending on other prevailing conditions, sotechniques to visualise the spectrum of NO species presentwill be very helpful. Clearly, different cells are likely to

respond in specific ways to NO, reflecting their owncomplement of active proteins, including signalling proteins.However, it may be that cellular specificity in NO responsesis also a consequence of variation in NO species (Murgia et al.,2002). Moreover, even though NO is diffusible, it might alsobe that cells differ, spatially and temporally, in their ability toreact with NO – such that in some cases NO effects may beconfined to specific cells or even specific microdomainswithin cells, an accepted concept for calcium signalling andone suggested recently for hydrogen peroxide and cyclicnucleotides (Neill et al., 2002c; Trewavas et al., 2002). Themobile nature of NO, and its chemical reactivity with variouscellular targets, mean that downstream effects of NO may bedirectly induced by interactions of NO with, for example, ionchannel proteins or proteins that regulate gene expression, orindirectly, that is, following interaction of NO with signallingproteins such as protein kinases, ion channels or secondmessenger-generating enzymes.

Although it is not always correct to assume that plant andanimal systems are the same, analogies with previously char-acterised animal signalling pathways can help to direct initialinvestigations into plant processes (see Wendehenne et al.(2001) for an excellent comparison of NO signalling in mam-malian and plant systems). NO signalling in mammalian cellstypically involves cGMP (cyclic guanosine monophosphate)as a second messenger, although there are also cGMP-independent pathways (Mayer & Hemmens, 1997; Bogdan,2001), and there are already data implicating cGMP signal-ling in plant responses to NO. Core components of mosteukaryotic signalling pathways usually include spatially spe-cific elevations in cytosolic Ca2+, either via release from intra-cellular stores, or via influx from the extracellular milieu. Thesecond major process in cell signalling is reversible proteinphosphorylation – signalling events frequently require proteinactivation by phosphorylation (or, less frequently, dephospho-rylation), followed by a subsequent return to a resting state viathe actions of constitutive (or sometimes inducible) proteinphosphatases. Thus far, there are only limited data regardingthese aspects of NO signalling in plants, although it seemslikely that they will also represent key components of NOresponses.

5.1 cGMP signalling

cGMP is a well-established second messenger molecule, thatis, a biologically active intracellular signalling molecule whoseconcentrations are transiently altered in response to anexternal stimulus. Typically, cGMP concentrations areincreased via enhanced activity of the biosynthetic enzymeguanylyl cyclase (GC), that synthesises cGMP from GTP(guanosine tri-phosphate) (Figs 5 and 6). Concentrations arereturned to resting values (and generally kept at low levels) bythe constitutive action of phosphodiesterases (PDE) (Fig. 5).In mammalian cells, many of the cellular effects of NO appear

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to be mediated by cGMP. NO activates cGMP production viainteraction with a soluble (and possibly also membrane-bound) form of GC; as NO is reasonably permeant, it reactsdirectly with the iron in the haem moiety of GC, inducing aconformational change that results in enzyme activation(Hancock, 1997). Such activation is transient, persisting onlyfor so long as NO is present. Thus the immediate cellulareffects of NO are relatively short-lived, as cGMP is rapidlydegraded by PDE.

Early reports of the existence of cyclic nucleotides in planttissues were not generally accepted, in part due to their verylow levels and the possibility of experimental artefacts (Newtonet al., 1999). However, unambiguous identification by massspectrometry has demonstrated unequivocally that theydo indeed occur in plant tissues, and various experimentalapproaches have recently generated exciting data showing thatthe intracellular concentrations of cyclic nucleotides alter inresponse to various stimuli, indicating that synthetic and deg-radative enzymes must be present (Newton et al., 1999;Trewavas et al., 2002). cGMP has been identified by massspectrometry in Zea mays (Janistyn, 1983) and Phaseolusvulgaris (Newton et al., 1984) and quantified in several species byradio-immunoassay (RIA). It is important to emphasise, how-ever, as do Newton et al. (1999), that RIA can generate false

positive data. Plant extracts are undoubtedly far more com-plex than mammalian plasma, and likely to contain com-pounds that can interfere in some way with the binding ofcGMP to an antibody. Thus some purification steps arerequired before quantitative analysis, and, ideally, RIA datashould be confirmed by comparison with data obtained by arigorous analytical technique that identifies and quantifiescGMP unequivocally. Generally, cGMP is found in plant tissuesin the pmol g−1 range, within the range found in mammaliancells, although there is considerable variation in the valuesreported (see Table 2). This is likely to reflect inherent technicaldifficulties and there is clearly a need for the developmentof appropriate technologies. The application of mass spectro-metry techniques such as electrospray ionisation, linked tocapillary and nanoflow liquid chromatography (LC-ESI-MS)may be particularly useful here. Indeed, Ehsan et al. (1998)have used this technology to quantify cyclic AMP (cAMP),purified via an immunoaffinity technique, in the pmol g−1

range in tobacco cells. cGMP appears similarly amenable toanalysis by such a method (S. J. Neill et al., unpublished) andit may be possible to use cGMP labelled with a heavy isotopeas an internal standard for isotope dilution studies. It will ofcourse, be essential to determine cGMP concentrations indiscrete tissues and cells (for example, stomatal guard cells, or

Fig. 5 Nitric oxide (NO) signal transduction.

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those specifically responding to pathogens). Even more chal-lenging, but potentially very revealing, will be the demonstra-tion of cGMP concentrations in specific microlocations, ormicrodomains, within cells (Trewavas et al., 2002). It seemslikely that various components of signalling pathways are notuniformly distributed within cells, and that specific stimuligenerate transient events in specific subcellular locations. Ifcellular responses depend on where within a cell such changesoccur, it will obviously be essential to visualise such changes.Honda et al. (2001) have recently described a moleculargenetic approach, in which they transformed cells with a constructcontaining a cGMP-sensitive protein (in this case cGMP-acti-vated protein kinase) linked to variants of green fluorescentprotein, to visualise intracellular cGMP via fluorescencemicroscopy. Using this approach, NO-induced changes incGMP were demonstrated in mammalian cells. Interestingly,NO-induced alterations in intracellular cGMP werevariable and appeared to reflect different activities of GCand PDE.

Several lines of evidence indicate the essential requirementfor cGMP synthesis and action during plant responses toNO. Pfieffer et al. (1994) used HPLC and RIA to quantifycGMP in spruce needles exposed for 10 min to gaseous NO(60 ppm). Basal levels of c. 0.015 µmol/g increased dramat-ically to c. 1.9 µmol/g, although these values do seem some-what out of step with those reported elsewhere (Table 2).Injection of recombinant rat NOS into tobacco leaves, ortreatment of tobacco suspension cultures with the NO donorGSNO, induced a rapid and substantial increase in cGMPcontent, from 5 to 10 to around 70–90 pmol/g within 1–2 h.Moreover, induction of PAL and PR-1 gene expression intobacco suspension cultures by GSNO was inhibited by theguanylyl cyclase inhibitors LY 83583 and ODQ. Although

one should always recognise the caveat that inhibitors arerarely (if ever?) specific (and in fact both LY 83583 and ODQare clearly not (Hausladen & Stamler, 1998; Feelisch et al.,1999)), treatment with a cell-permeable analogue of cGMP,8-Bromo-cGMP, was able to counteract the inhibitory effectsof LY 83583, strengthening the case for cGMP involvement.Moreover, treatment with 8-Br-cGMP alone was sufficient toinduce the expression of both PR-1 and PAL. In Arabidopsissuspension cultures, NO-induced PCD was inhibited byincubation with ODQ, and relieved by coincubation with8-Br-cGMP. Treatment with 8-Br-cGMP alone however, didnot induce PCD, indicating that cGMP was required, but notsufficient, for NO induction of cell death (Clarke et al.,2000). A similar requirement for cGMP during ABA- andNO-induced stomatal closure has been found for pea (Neillet al., 2002a) and Arabidopsis (R. Desikan et al., unpublished).Again, treatment with 8-Br-cGMP alone was not able tomimic the effects of ABA and NO. Thus, it seems that cGMPis a sufficient intracellular mediator for some signalling path-ways, but for others, additional intracellular signals are alsorequired. This is underscored by the apparent contradictionbetween those data showing that stomatal opening in Com-melina communis induced by auxin, kinetin or a natriureticpeptide require the synthesis and action of cGMP (Cousson& Vavasseur, 1998; Pharmawati et al., 1998a,b) and thosedemonstrating the same requirement for ABA- and NO-induced stomatal closure in pea and Arabidopsis (Neill et al.,2002a; R. Desikan et al., unpublished). In fact, in our laboratory,8-Br-cGMP alone induces neither stomatal opening norclosure in Arabidopsis, but does counteract the inhibition ofIAA-induced stomatal opening and of ABA or NO-inducedclosure by the GC inhibitor ODQ. This implies that IAA,ABA and NO activate cGMP synthesis and at least one other

Table 2 Cyclic GMP (cGMP) concentrations in plants

Resting levels, Basal concentrations

Stimulus, elevated concentrations Tissue Method; comment Reference

3.3 pmol/g Bean RIA; identified by Newton et al. mass spectrometry (1984)

0.07 pmol/g GA, 0.28 pmol/g after 2 h; Barley aleurone cells RIA; inhibited by Penson et al.lowered after 4 h LY 83583 (1996)

15 pmol/g Anaerobiosis, Rice shoots RIA Reggiani et al. (1997)40 pmol/g 38 pmol/g Rice roots

80 pmol/g0.015 µmol/g NO gas (60 ppm) Spruce seedlings, HPLC/RIA, good Pfieffer et al. (1994)2.8 nmol/g 1.9 µmol/g Potato agreement Pfieffer et al.

29 nmol/g (1995)

0.25 pmol/g Plant natriuretic peptide Maize root stele RIA, LY 83583 had no Pharmawati et al. analogue with PDE inhibitor effect (1998a)1.3 pmol/g

5–10 pmol/g Rat NOS, 90 pmol/g after 2 h Total leaf RIA Durner et al. (1998)< 5 pmol/g GSNO, 70 pmol/g after 0.5 h Tobacco cells

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signalling event that is required for stomatal opening or clo-sure, respectively. In the case of NO responses, the molecularnature of such additional signals also awaits elucidation; cal-cium is a likely candidate.

The transient increases in cGMP content induced by NO,and inhibition of NO responses by GC inhibitors, mean thatthe enzymes required to synthesise cGMP are present, and insome cases, rapidly activated, and that the (presumably phos-phodiesterase, PDE) enzymes that effect metabolic inactiva-tion, are also present, being either constitutively active orsimilarly activated. In fact, cGMP-PDE enzyme activity hasbeen found in plant tissues (Newton et al., 1999) and severalpotential PDE genes exist in the Arabidopsis genome (TheArabidopsis Genome Initiative, 2000), although functionalcharacterisation of a plant cGMP-PDE has not yet been achieved.The reported effects of ViagraTM (Pfizer, UK) in plants suggestthat cGMP-PDE activity is present and terminates the effectsof NO (Leshem, 2001); ViagraTM, sildenafil citrate, the world’sfastest-selling drug, used clinically to treat erectile dysfunc-tion, is a cGMP-PDE inhibitor (Fricker, 2001). Moreover,

although cGMP synthesis has been demonstrated in plantextracts (Newton et al., 1999) no obvious plant GC has yet beencloned. A comparison with the situation of adenylyl cyclase(AC) might be useful here. A plant AC gene has recently beencloned from maize pollen (Moutinho et al., 2001). This AChas only limited sequence homology with previously clonedAC enzymes and it is likely that the AC enzyme family is arather diverse one, such that database searching by simplesequence homology may not be fruitful. In another example ofthe potential complexity of cyclic nucleotide metabolism, aDictyostelium gene with homology to mammalian soluble AC wasrecently characterised that, in fact, encoded a protein possessingGC activity. Although orthologues to this gene are present inmammals, they are not, apparently, in Arabidopsis (Roelofset al., 2001). A novel gene encoding a protein with GC activity,AtGC1, has recently been characterised by Ludidi & Gehring(2003). It will be interesting to determine the role of this enzymein cell signalling generally, and NO synthesis in particular.

5.2 cADP-ribose (cADPR) and calcium

Intracellular calcium can be stored in various cellularlocations, release from which is predicated by the presence ofspecific calcium channel proteins that recognise and bindsecond messengers such as IP3 and cADPR. cADPR (Fig. 6)is a well-characterised calcium-mobilising second messengerin animal cells that activates calcium release from a discretesubset of membrane vesicles. cADPR synthesis in animal cellsis commonly activated by NO, such activation beingmediated by cGMP, potentially via activation of a cGMP-dependent kinase (Wendehenne et al., 2001). cADPR canalso induce Ca2+ release from plant endoplasmic reticulumand vacuoles (Allen et al., 1995; Leckie et al., 1998). Intobacco cells, cADPR was able to mimic NO induction ofPR-1 and PAL gene expression, and the cADPR effects wereinhibited by Ruthenium Red, an inhibitor of intracellularCa2+ release (Durner et al., 1998). In addition, 8-Br-cADPR,an antagonist of cADPR, inhibited PR-1 gene expressioninduced by recombinant NOS (Klessig et al., 2000).

NO induces stomatal closure and is an essential intermediatein ABA-induced stomatal closure (Garcia-Mata & Lamattina,2001; Garcia-Mata & Lamattina, 2002; Neill et al., 2002a).Calcium is a core component of stomatal ABA signalling pathways,with a full response to ABA being calcium-dependent, but apartial response likely to be calcium-independent (Webbet al., 2001). It is not surprising then, that NO-induced sto-matal closure requires calcium (Garcia-Mata & Lamattina, 2001).The sources of calcium, however, remain to be determined.Both calcium influx from the cell exterior and calcium releasefrom intracellular stores are required for ABA effects(MacRobbie, 2000). In addition, it appears that intracellularcalcium can be released from at least two separate sources –vesicles sensitive to IP3 as well as those sensitive to cADPR.cADPR induces partial stomatal closure in Commelina

Fig. 6 Cyclic GMP and cyclic ADP-ribose.

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communis and treatment with either a cADPR antagonist, ornicotinamide, a potential inhibitor of cADPR synthesis,reduced ABA-induced stomatal closure and ABA-inducedvacuolar ion efflux in C. communis (Leckie et al., 1998;MacRobbie, 2000). In pea, nicotinamide inhibited both ABAand NO-induced stomatal closure (Neill et al., 2002a). Zocchiet al. (2001) recently reported that ABA stimulates cADPRsynthesis in the sponge Axinella polyploides, leading to the sug-gestion that ABA/cADPR signalling may have originated inan evolutionary precursor of Metazoa and Metaphyta. So far,though, cADPR has not yet been identified unequivocally inplant cells nor has its biosynthetic enzyme, cADPR cyclase,been cloned. However, there is good evidence that cADPRmediates ABA-induced gene expression in tomato and thatABA can induce increases in cADPR in tomato and Arabidop-sis (Wu et al., 1997; J. Sanchez, pers. comm.). Moreover, inArabidopsis transformed with an Aplysia cADPR cyclase genelinked to a sterol-inducible promoter, sterol treatmentinduces partial stomatal closure in the absence of ABA ( J.Sanchez & N-H. Chua, pers. comm.). These data would beconsistent with ABA inducing NO synthesis in guard cells,leading to subsequent synthesis of cGMP and cADPR.

5.3 NO and cGMP-activated protein kinases/protein phosphatases

A common downstream target of cGMP in mammalian cellsis cGMP-activated protein kinase (Wendehenne et al., 2001).Although plants contain an impressive array of proteinkinases, thus far a cGMP-activated PK has not yet beenidentified. However, NO has been shown to activate PKs inArabidopsis and tobacco (Clarke et al., 2000; Kumar & Klessig,2000; Fig. 5). It is not known if the effects of NO were direct,or via activation of other signalling molecules that subsequentlyactivate the PKs. In fact, the NO-activated PK in tobacco wasidentified as SIPK, an SA-activated mitogen activated proteinkinase (MAPK), and SA was required for NO activation ofSIPK, as it was not activated in the NahG mutant that has areduced SA content (Kumar & Klessig, 2000). Moreover, atleast in tobacco, NO induces increases in endogenous SA(Durner et al., 1998). The NO-activated PK in Arabidopsishas not yet been identified, but did have the characteristics ofa MAPK (Clarke et al., 2000). Whether or not activation ofthese kinases is mediated by cGMP remains to be seen.

NO synthesis and signalling also involves regulation viaprotein phosphatases. Treatment of soybean suspension cul-tures with cantharidin, a protein phosphatase 2 A inhibitor,led to increased NO synthesis (Delledonne et al., 1998). Bycontrast, NR-mediated NO synthesis in spinach was shownto be inhibited by cantharidin (Rockel et al., 2002). In Arabi-dopsis, the ABA-insensitive mutants abi1–1 and abi2–1 inwhich different PP2C genes are mutated, do synthesise NOin response to ABA, but their stomata are unable to close inresponse to treatment with NO (Desikan et al., 2002).

5.4 Cyclic nucleotide gated ion channels (CNGCs)

CNGCs are defined functionally as ligand-gated ion channelsthat are activated by cyclic nucleotide binding to the channelprotein, but there also exist channels that may be gated byother stimuli such as voltage but whose activity is modulatedby cyclic nucleotides, as well as those whose activity can beaffected by phosphorylation via cyclic nucleotide-dependentprotein kinases (Leng et al., 1999). Several cyclic nucleotide-gated ion channels (CNGCs) have now been cloned fromplants (Trewavas et al., 2002), including those responsive tocGMP (Gaynard et al., 1996; Schuurink et al., 1998; Kohleret al., 1999; Leng et al., 1999; Arazi et al., 2000). InArabidopsis, the CNGC gene family contains at least sixmembers, encoding channel proteins with the characteristicsix potential membrane–spanning domains and a putativecyclic nucleotide binding domain (Kohler et al., 1999). Theseproteins also contain a functional calmodulin binding site(Kohler & Neuhaus, 2000), indicating the potential for cross-talk between calcium and cGMP signalling pathways.Mutation of the Arabidopis AtCNGC2 gene results in thednd1 (‘defense, no death’) mutant. This mutant displayselevated SA content, salicylic acid-dependent constitutivedefence responses and loss of the Hypersensitive Response(HR) (Clough et al., 2000), demonstrating a physiologicalfunction for this CNGC. It is interesting to note that, at leastin tobacco, NO induces SA biosynthesis and thus responsesdownstream of SA (Durner et al., 1998; Klessig et al., 2000),suggesting the possibility that one normal consequence ofAtCNGC2 channel activity might be to repress SA synthesis.When expressed in yeast or human cells, AtCNGC2 mediatesflux of K+ and Ca2+ ions in the presence of either cAMP orcGMP (Leng et al., 1999), but its channel and activationcharacteristics in planta are not yet characterised. Theexpression of AtCNGC2 is developmentally regulated inArabidopsis, being increased during sensecence (Kohler et al.,2001). Determination of the expression profile of all membersof the CNGC family activated by cGMP is a clear researchpriority. For example, some stomatal responses to NO may beeffected through cGMP-mediated activation of CNGCs.

5.5 cGMP-independent effects of NO

Although cGMP-independent effects of NO are known, thebiochemical mechanisms are not completely understood(Hausladen & Stamler, 1998; Bogdan, 2001; Wendehenneet al., 2001) with novel processes being characterised recently,for example nitrolinoleate-mediated NO signalling (Limet al., 2002). The chemistry of NO means that transitionmetal- (e.g. iron, copper, zinc) and thiol-containing proteinsare major cellular targets (Beligni & Lamattina, 2001;Bogdan, 2001; Wendehenne et al., 2001). Similar to itsinteraction with GC, NO can complex with iron in otherhaem and iron-containing proteins. Thus, NO inhibits

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tobacco aconitase, an iron-sulphur-containing enzyme thatcatalyses the isomerisation of citrate to isocitrate (Navarreet al., 2000). In addition to the subsequent effects onmetabolism (aconitase is a component of the Krebs cycle), itis possible that NO converts the cytosolic form of aconitaseinto IRP1 (iron-regulatory protein), a protein that is involvedin cellular iron homeostasis. In animal cells IRP1 inhibits thetranslation of mRNA encoding the iron-binding proteinferritin by binding to iron-responsive elements in the 5′untranslated region (Wendehenne et al., 2001; Murgia et al.,2002). This would lead to a reduction in cellular ferritincontent, resulting in an increase in free iron that maysubsequently lead to generation of ROS such as the hydroxylradical via the Fenton reaction, with consequent cell death(Wendehenne et al., 2001). However, mechanisms of ironhomeostasis may differ between plants and animals, withferritin content being regulated transcriptionally in plants.Murgia et al. (2002) found that NO induced the accumu-lation of both ferritin mRNA and protein in Arabidopsis,acting through the IDRS (iron-dependent regulatory sequ-ence) present in the ferritin promoter. NO also inhibits thehaem-containing enzymes catalase and peroxidases, withpotential knock-on effects on ROS levels and xylem develop-ment (Ferrer & Barcelo, 1999; Clark et al., 2000; Barcelo et al.,2002).

It is well-known that NO can interact (probably via NO+)with reactive amino acids such as cysteine and tyrosine in pro-teins and with thiol groups present in other molecules such asthe ubiquitous redox–sensitive and potentially regulatorytri-peptide glutathione (Jia et al., 1996; Wendehenne et al.,2001). Thiol modification by ROS such as hydrogen peroxideis already recognised as a potential signalling mechanism inplants (Neill et al., 2002b). Because thiol residues and disul-phide bridges can be important for tertiary protein structure,reversible modification of thiol groups and disulphide bondsmay have important consequences for protein activity, analo-gous to protein modifications via reversible phosphorylation.Protein thiol residues have recently been described as poten-tial ‘nanotransducers’ (Schafer & Buettner, 2001); the exist-ence of several forms, generated by reaction with ROS andreactive nitrogen species (RNS) that might compete for thesame thiol group, allowing ‘graded’ activity responses asopposed to ‘on or off ’ switching effected by phosphorylation(Cooper et al., 2002). S-nitrosylation of proteins and glutathionehas been demonstrated in vitro, formation of S-nitrosothioladducts shown to alter protein activity and some endogenousS-nitrosylated proteins and S-nitrosothiols have been demon-strated (e.g. haemoglobin, Jia et al. (1996); and GSNO, Hogg,(2000)). However, to date no endogenous S-nitrosylated pro-teins have been characterised in plants, although the recentdevelopment of a sensitive proteomic approach to identifyendogenous S-nitrosylated proteins may well be useful here(Jaffrey et al., 2001). In this study, various S-nitrosylated pro-teins were detected and shown to be absent in knock-out mice

in which neuronal NOS was deleted, indicating a physiolog-ical role for S-nitrosylation. As with high molecular weightS-nitrosothiols, GSNO has not yet been identified in plants;analysis by LC-MS may be possible (Kluge et al., 1997).GSNO is often used as an NO donor, but may induce biolog-ical responses via mechanisms not involving NO release, suchas trans-nitrosation of thiol groups present in proteins,thereby modifying their activity (Hogg, 2000).

5.6 NO metabolism

The biological effects of RNS such as NO and GSNO makeit likely that endogenous mechanisms exist to remove them.Stamler’s group recently characterised an enzyme activityfrom Escherichia coli that degrades GSNO (Liu et al., 2001).The enzyme was identified by mass spectrometry asglutathione-dependent formaldehyde dehydrogenase (GS-FDH). In fact, the GSNO reductase (GSNOR) activity ofthis protein was much greater than its GS-FDH activity, andalso identified in yeast and mammalian cells. Knock-out miceand yeast, in which the GSNOR gene was deleted, had muchreduced GSNO-catabolising capacity, and accumlulatedprotein S-nitrosothiols and some GSNO. In addition, theGSNOR-deficient yeast was much more susceptible tonitrosative stress. The endogenous amounts of GSNO werevery low in wild type mouse macrophage cells but GSNOreadily accumulated in the culture medium of interferon–treated cells (interferon activates NOS) supplied withglutathione. Thus the role of GSNOR may be to maintainlow endogenous levels of GSNO and thereby preventexcessive formation of S-nitrosylated proteins to damaginglevels, presumably by trans-nitrosation reactions with GSNO.GS-FDH genes have been cloned from various plant species(Sakamoto et al., 2002) and now the Arabidopsis GS-FDHprotein has, like its microbial and mammalian counterparts,been shown actually to possess GSNOR activity (Sakamotoet al., 2002). Moreover, the Arabidopsis gene was able tocomplement the yeast GSNOR knock-out mutant. Expres-sion of the Arabidopsis GSNOR is constitutive and relativelyhigh, in keeping with a potential role in maintaining lowendogenous levels of GSNO. No doubt subsequent studiesusing a transgenic approach to manipulate the level ofGSNOR activity will reveal much more about its function.

Other NO metabolising mechanisms have been character-ised that may have relevance for plant–pathogen interactions.NO generation by mammalian cells represents a potentdefence mechanism against bacterial pathogens, as NO istoxic to bacteria at high concentrations (possibly via conver-sion to peroxynitrite). Several groups have shown that bacte-rial flavohaemoglobin (HMP) serves a protective role inbacteria such as Salmonella typhimurium and Escherichia coliagainst nitrosative stress that might be imposed within thehost environment. These bacterial HMP proteins possessNO-dioxygenase activity, converting NO to nitrite and

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nitrate (Crawford & Goldberg, 1998; Gardner et al., 1998;Hausladen et al., 1998). HMP gene expression is inducible byNO, via a mechanism that in E. coli involves NO interactionwith the iron-sulphur clusters present in the global transcrip-tion factor FNR. In the absence of NO, FNR binds to thepromoter regions of hmp, repressing gene expression. Follow-ing complex formation with NO, FNR is inactivated, andthus HMP gene expression de-repressed (Ramos et al., 2002).In the phytopathogen Erwinia chrysthanthemi, flavohaemo-globin HmpX was described as a virulence determinant(Favey et al., 1995). It may be that HmpX is required for vir-ulence because it mediates bacterial detoxification of host-generated NO. Differing sensitivities to NO have also beenreported for other phytopathogenic bacteria (Alamillo &Garcia-Olmedo, 2001). It will be interesting to see if otherphytopathogens require NO-metabolising enzymes for viru-lence; if so, development of inhibitors of bacterial flavohaemo-globins may represent a novel approach to plant protection.

5.7 NO transport

Although NO is a reasonably diffusible gas, it is possible thatNO precursors or NO-adducts could act a ‘stores’ of NO, ortransport forms operating over short or long distances,analogous to flooding-induced transport of the ethyleneprecursor ACC from roots to shoots in flooded plants(Jackson, 2002). For example, in animals the presence of S-nitrosohaemoglobin in circulating blood led to the suggestionthat it might serve as a source of circulatory NO (Jia et al.,1996). This is now considered unlikely, but instead, evidencehas been presented that nitrite is present in circulating bloodand can serve as a delivery source of NO (Gladwin et al.,2000). As nitrite can clearly serve as a precursor to NO inplant cells, including guard cells that are continually exposedto the xylem stream (Desikan et al., 2002), it is a possibilitythat nitrite can serve as a mobile source of NO. There is alsothe possibility that GSNO, in either phloem or xylem, isanother mobile source of NO in plants. There is then theintriguing possibility that these NO-precursors may bedelivered from flooded roots to shoots, generating NO in theshoots and inducing the stomatal closure that is observed butnot attributable to ABA (Jackson, 2002). Plants also containhaemoglobin genes (Hunt et al., 2001), so it is possible thatplant haemoglobins can form reversible NO complexes.

5.8 NO interactions with other signalling pathways

A reductionist approach that evaluates the biological roles ofNO and the underlying signalling mechanisms in isolation isinevitably a deficient one. Clearly NO can interact with othersignals either directly, for example, with superoxide to formperoxynitrite (see section 2 above) or with other signallingpathways to direct cellular activities. A comprehensive,holistic consideration of the interactions and signalling

cross-talk involving NO is outside the scope of this review.However, it is pertinent to make a few observations. Inparticular, much experimental data indicate that generation ofROS such as hydrogen peroxide (potentially arising fromsuperoxide generated via NADPH oxidase; Neill et al.,2002b) appears to be a common companion to NOgeneration. Thus, both ROS and NO are generated inresponse to pathogen challenge (Durner & Klessig, 1999) aswell as in stomatal guard cells in response to ABA (Neill et al.,2002a). H2O2 is made in response to many biotic and abioticstimuli (Neill et al., 2002b) and it may well be that so too isNO, and that subsequent responses depend, in part at least,upon interactions between H2O2 and NO, as outlined in theprevious section. This is well illustrated by the data ofDelledonne et al. (2001) described in section 4.7 above. It isalso possible that NO can inhibit superoxide generation(Caro & Puntarulo, 1998). It is also worth pointing out thatboth H2O2 and NO activate potential MAPKs and changesin intracellular calcium, further points of cross–talk forinteraction of NO with H2O2 and other stimuli. Interactionsbetween ABA and NO during stomatal closure in V. faba werehighlighted by Garcia-Mata & Lamattina (2002). Clearly NOcan, and does interact with many signals, but a complete ‘NOsignalling map’ will take some time to complete.

5.9 NO and gene expression

It is apparent from the previous sections that a growingnumber of physiological and developmental responses to NOhave been demonstrated in plants. Thus there must inevitablybe changes in the spectrum of gene expression followingexposure to NO. In fact, NO induction of gene expressionwas first shown during plant–pathogen interactions. Intobacco, infection of resistant plants with TMV inducedincreased NOS activity (Durner et al., 1998) and injection ofrecombinant NOS or exposure to the NO donors GSNO orSNAP induced the expression of PAL and PR-1 genes(Durner et al., 1998). PR-1 gene expression was also inducedby TMV infection, an effect suppressed by coinjection withthe NOS inhibitor -NAME, providing evidence for theendogenous mediation of TMV-induced gene expression byNO (Klessig et al., 2000). Delledonne et al. (1998) showedthat SNP induced the expression of PAL and CHS in soybeansuspension cultures. Subsequent to this, there have been onlylimited reports on NO-induced gene expression. A.-H.-Mackerness et al. (2001) showed that NO scavenging withPTIO or inhibition of NOS by L-NAME inhibited UV-Binduction of CHS gene expression in Arabidopsis plants.Direct exposure to NO, via treatment with either GSNO orSNAP, also induced CHS gene expression, suggesting thatendogenous NO mediates UV-B induction of CHS geneexpression. Murgia et al. (2002) have shown that NO inducesferritin mRNA accumulation in Arabidopsis plants andsuspension cultures. Moreover, NO is required for iron

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induction of ferritin transcript accumulation, as removal ofNO with cPTIO is inhibitory. A recent microarray studyidentified a large number of genes that were induced by NO(applied via the NO donor NOR-3) in Arabidopsis suspensioncultures (Huang et al., 2002). In this work, particular attentionwas paid to the AOX1a gene, encoding one member of thesmall enzyme family of alternative oxidases (see section 4.7above). AOX1a gene expression was induced within 2 h bytreatment with NOR-3, such induction being suppressedstrongly by removal of NO with cPTIO, confirming that theeffects of NOR-3 were indeed due to NO generation.Furthermore, AOX1a expression was induced within 1 hfollowing exposure of plants to gaseous NO. Other genestransiently induced by NO in suspension cultures includedseveral pathogenesis-related proteins and antioxidant genesincluding peroxidases and glutathione-S-transferases, as wellas some encoding likely signalling proteins (Huang et al., 2002).

What are the potential mechanisms by which NO inducesgene expression? The induction of many genes (c. 5% of thoseon the array) by NO (Huang et al., 2002) suggests a commonmechanism for at least some of these genes. Thus far, though,no bioinformatic data are available with respect to the pres-ence of common potential regulatory cis elements in the pro-moter regions of the genes analysed by Huang et al. (2002).On the other hand, Murgia et al. (2002) showed that NOinduction of ferritin gene expression was mediated via theIRDS sequence in the ferritin promoter.

Given that NO can change protein conformation in severalways, including S–nitrosylation, interaction with iron inhaem groups and reactions with iron and other transitionmetals such as zinc, commonly found complexed with histi-dine and cysteine residues in zinc finger motifs often presentin transcription factors, it is possible that NO alters transcrip-tional profiles directly, by diffusing into the nucleus and

activating or inactivating transcription factors (Fig. 7). On theother hand, as NO also activates signalling processes includ-ing SA production, cGMP synthesis, calcium fluxes, andreversible protein phosphorylation, it is also likely that NOeffects on transcription are mediated via modulation of tran-scription factor activity in signalling cascades requiring suchprocesses (Fig. 7). For example, NO induction of PR-1 geneexpression in tobacco required SA synthesis and action,whereas induction of PAL gene expression did not (Durneret al., 1998). Furthermore, although PR-1 gene expressionwas induced by cGMP and cADPR, NO induction of PR-1expression was not completely suppressed by an antagonist ofcADPR, indicating that PR-1 expression is mediated by bothcGMP-dependent and independent pathways (Klessig et al.,2000). In Arabidopsis suspension cultures, AOX1a geneexpression was induced by NO much earlier than was SAbiosynthesis, and was still induced in various mutants withimpaired SA signalling or reduced SA content (Huang et al.,2002).

Conclusion

Recent years have seen a huge increase in research activitiesaimed at elucidating the biological roles of NO in plants andthe underlying signalling mechanisms. There can be no doubtthat NO is an endogenous metabolite, but its biosyntheticorigins still remain to be completely characterised. CertainlyNO can be synthesised from nitrite via nitrate reductase, butthe presence of an enzyme similar to mammalian NOS hasnot yet been confirmed. Other sources of NO, bothenzymatic and nonenzymatic, are also possible. Identificationand subsequent manipulation of NO biosynthetic enzymesand the encoding genes is a research priority. Application ofNO via NO donors indicates that NO can induce variousprocesses in plants, such as PCD, stomatal closure, and rootgrowth, although it should be borne in mind that differentNO donors may release different molecular species of NOthat could have distinct effects. Nevertheless, pharmacologicalmanipulation using NO scavengers and inhibitors of NOsynthesis does indicate very strongly that endogenous NOmediates various responses to developmental and externalstimuli. There is now a requirement for methods to assay NOsynthesis and quantify its release from cells. Intracellularsignalling responses to NO involve generation of cGMP andcADPR and elevation of cytosolic calcium, but in many casesthe precise biochemical and cell biological nature of theseresponses is yet to be detailed. There is a clear need for thedevelopment of techniques to identify, visualise and quantifycGMP and cADPR in plant cells, and to clone the genesrequired for their synthesis and degradation, such thattransgenic approaches can be used for functional analyses.Similarly, PKs and PPs, transcription factors, ion channelsand other signalling proteins activated or repressed by NOawait identification and characterisation. Despite the already

Fig. 7 Nitric oxide (NO) and gene expression. NO might interact directly with transcription factors (TFs) modulating activity and thus gene expression. Alternatively, NO can affect transcription by altering the activity of signalling pathways ultimately affecting transcription factors.

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exponential growth in plant NO research, there is a need formuch more – given the essential roles of NO in plant growthand development, such research efforts will surely be justified.

Acknowledgements

We are grateful to all those colleagues who provided reprints,preprints and unpublished data for this review.

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